Jasminum Officinale Against Oral Pathogens in Ulcer Treatment

ISSN: 2455-2631 © June 2019 IJSDR | Volume 4, Issue 6

Determination of Antibacterial Activity of Leaf Extract of Jasminum officinale Against Oral Pathogens in Ulcer Treatment

1Kanchan N. Mangtani 2 Dr. Shubhangi S. Morey 3Dr. Umesh K. Bhalekar

1Student 2Clock hour basis (C.H.B) 3Head of Department Department of Microbiology Shri R.L.T. College of Science, Akola 444002

Abstract: From ancient times, plants have been used in traditional medicines for treatment of different ailments. Medicinal plants is one of the richest bio resources for traditional and folk medicines till date. Jasmine is botanically known as Jasminum officinale or Jasmininie and belongs to the olive family of Oleaceae. Literature report suggest that Jasmine is analgesic, antidepressant, antiseptic, expectorant, aphrodisiac, sedative, stomachic, diuretic, depurative, astringent, stimulating, anti-oxidizing, anthelmintic and anti-inflammatory in nature. The objective was to study antibacterial activity of Jasminum officinale extracts against mouth ulcer causing organisms. The antibacterial activity has been studied against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis & Enterococcus faecalis by agar well diffusion method. Leaves extract of J. officinale give effective results against oral pathogens causing mouth ulcer. Acetone and Ethanol extracts displayed a good antibacterial activity. The phytochemical studies revealed presence of Carbohydrates, Proteins, Steroids, Alkaloids, Flavonoids, Phenols, Saponins, Glycosides and Tannins. J. officinale may prove to be effective medicine for the treatment of ulcer. Keywords: Jasminum officinale, Antibacterial activity, Phytochemical analysis

I. Introduction From ancient times, plants have been used in traditional medicines for treatment of different ailments. Medicinal plants is one of the richest bio resources for traditional and folk medicines till date. Around 20,000 medicinal plants have been recorded in India. Only 7,000 - 7,500 plants are used for curing different diseases. The antimicrobial potential and antioxidant activity of plants have attracted the attention of scientific community from ancient times. This had lead to increase in the interest of natural substances exhibiting antimicrobial and antioxidant properties. (1) The treatment of disease began long ago with the use of herbs. Herbs became the sources of many important drugs due to its wide range of therapeutic and pharmacological effects. (2) Indian system of medicines comprises of Ayurveda, Unani, Siddha, Homeopathy, Naturopathy and Yoga. Each of which uses the herbal constituents in some or the other form, crude drug is not so effective because they have not been tested for efficacy according to rigid pharmacological standards. As the constituents derived from the medicinal plants proved to cure the human disorders they isolated and used for their pharmacological action. Jasmine is botanically known as Jasminum officinale or Jasmininie and belongs to the olive family of Oleaceae. Jasmine is analgesic, antidepressant, antiseptic, expectorant, aphrodisiac, sedative, stomachic, diuretic, depurative, astringent, stimulating, anti-oxidizing, anthelmintic and anti-inflammatory in nature. Furthermore, there are other numerous advantages this amazing plant offers to humanity. These benefits have been attributed to its phytochemical, medicinal and pharmacological properties. (3)

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Jasminum officinale indicated the presence of alkaloids, flavonoids, tannins, terpenoids, glycosides, steroids, essential oil and saponins. Studies revealed that the plant exerted antimicrobial, insecticidal, antioxidant, antifertility and dermatological effects. (2)

Fig 1. Jasminum officinale (3) Classification of Jasminum officinale (2) Kingdom: Plantae Subkingdom: Viridiplantae Division: Tracheophyta Class: Magnoliopsida Order: Lamiales Family: Oleaceae Genus: Jasminum Species: Jasminum officinale Health Benefits of Jasminum officinale The aroma of jasmine is calming and soothing without being soporific and is indicated for depression and stress. It is indicated for sensitive skin conditions too. Jasmine also has a reputation as an aphrodisiac and used for all kinds of sexual problems. Jasminum is used to treat skin problems, the leaf juices can be applied to clear up corns and treat mouth ulcerations, the anti-secretary and anti-oxidant components of Jasminum may also treat peptic ulcer. Jasminum also produces an antibiotic effect upon typhoid fever and staph infections, they stressed that jasmine oil may serve as a main stream antibiotic treatment, the juice of the leaf is applied to corns and ear discharges, the leaves and the barks contain salicylic acid and are used as analgesic, febrifuge, etc. The roots are used in the treatment of ringworm, while the flowers are aphrodisiac, antiseptic, antispasmodic, and tonic. One of the uses of J. officinale in urinary infections and diuretic the leaves of stem, bark, and root of Jasminum has demonstrated detectable antibacterial activity against many microorganisms. (4) 1) Mouth ulcers Mouth ulcers are painful sores on the inside lining of the mouth. They usually develop on the inside of the lips and cheeks and on the underneath and edge of the tongue. Medicines from a pharmacist can reduce the pain and help mouth ulcers to heal. Mouth ulcers include sores, lesions, abrasions, laceration or any open break in the mucosa of the lips, mouth or tongue. Mouth ulcers are also called stomatitis and are a symptom of a variety of mild to serious diseases, disorders and conditions. Mouth ulcers can result from infection, vitamin deficiencies, trauma, inflammation, malignancy and other diseases and abnormal processes. (5)

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2) Causes The exact reason of mouth ulcers developed is not yet clearly defined. Approximately 40% of people who get mouth ulcers have a family history of the same. In some cases, the ulcers are related to diseases. These include Injury from badly fitting dentures, harsh brushing of teeths, etc. Changes in hormone levels. Some women find that mouth ulcers occur just before their periods. A lack of iron or a lack of certain vitamins (such as vitamin B12 and folic acid) may be a factor in some cases. Rarely, a food allergy may be the cause. Stress is said to trigger mouth ulcers in some people. Some medicines can cause mouth ulcers. Examples of medicines that can cause mouth ulcers are: nicorandil, ibuprofen etc. Mouth ulcers are more common in people with Crohn's disease, coeliac disease, HIV infection etc. (6) 3) Bacteriology In the mouth there are many good and bad micro-organisms and bacteria, which now have access to the wound surface and produce toxins which in turn allows further cell death causing the ulcer to get larger. Also, at this stage the bacteria lining the ulcer. This situation continues till the causative agent is gone and the body’s immune system comes up with the solution and the bad bacteria are compressed. How long this takes depends on many factors. Staphylococcus, Pseudomonas, Bacillus, E.coli, Enterococcus and Candida species are an important component normal flora of the Oropharynx. (7)

Bacteria: 1. Escherichia coli. 2. Pseudomonas aeruginosa. 3. Staphylococcus aureus. 4. Bacillus subtilis. 5. Enterococcus faecalis

Fungi 1. Candida albicans. II. Aims & Objective To prevent side effects from antibiotics on human body and see importance of natural antibiotics (Plant extracts) has been undertaken with following aims & objectives.  Selection and collection of Jasminum officinale leaves from different area in Akola region  Extraction of plant leaves material.  Phytochemical analysis of Jasminum officinale leaves extract.  Collection of ulcer samples from the patients having mouth ulcer.  Isolation and identification organisms.  To check the antibacterial activity of Jasminum officinale extract against isolated organisms. III. Materials and Methods A) Preparation of plant leaves extract i) Collection of Plant material Fresh leaves of Jasminum officinale were collected from Akola city. The collected plant leaves were washed with water to remove other undesirable material. The leaves were spread on the tray and allowed to dry at room temperature under shade for several days. The air-dried leaves of Jasminum officinale were crushed. The dried leaves were grinded into powder using an electrical blender. Fine powder was stored in airtight container.

ii) Preparation of extract using soxhlet apparatus (9)

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Plant extract were prepared by using Soxhlet apparatus. For this 30gm of plant leaves powder was wrapped in muslin cloth and loaded in the thimble of the soxhlet apparatus. The solvent was added in the round bottom flask. The solvent used were ethanol and aqueous. The boiling point of ethanol is 780C. The temperature was maintained by using heating mantle for recycling of solvent. It was continuously extracted till 3 cycles complete. The extract was filtered through Whatman No. 1 filter paper. The Filtrate were collected in sterilized iodine flask and stored at 50C in the refrigerator until futher use. Similarly, the same extraction process is carried out using aqueous and acetone solvent

Fig 2. Powder of Jasminum officinale leaves Fig 3. Extraction of leaves by Soxhlet apparatus B) Sampling and isolation of oral pathogens i) Sampling The samples were collected from different patients suffering from mouth ulcer by using swab. ii) Isolation and identification of organism The collected samples were inoculated on selective media. HiCrome UTI Agar, Eosin Methylene Blue Agar (EMB), MacConkey Agar, Baird Parker Agar (B.P) and Nutrient Agar plates were inoculated by swabbing and incubated at 370C for 24 hrs. Next day colonies which were appeared on Nutrient Agar were further identified by using selective media. Cetrimide Agar for Pseudomonas aeruginosa and Luria-Bertani Agar (LB) for Bacillus subtilis. The colonies of Escherichia coli and Staphylococcus aureus appeared on their selective media such as EMB agar, B.P. Agar respectively. On UTI Agar green colour colonies appeared. These isolates were also identified by Gram staining and biochemical test such as sugar fermentation test, IMViC and other test. After identification of bacteria these isolates were inoculated on Nutrient Agar as a pure culture. Then the antibacterial activity of Jasminum officinale was tested against these isolated organisms. C) Phytochemical analysis of Jasminum officinale leaves extract (9) The Phytochemical analysis of plant extracts were carried out by using standard qualitative methods for identification of active chemical constituents such as sterol, alkanoids, flavonoids, terpenoids etc., were identified by characteristics colour change development by standard procedures. The ethanolic, aqueous and acetonic extract of the leaves of Jasminum officinale were used for phytochemical analysis. i) Test for carbohydrates Benedict’s test To 0.5ml of the leaves extract, 5ml of Benedict’s reagent was added and boiled for 5 minutes. Appearance of red precipitate showed the presence of reducing sugar. ii) Test for Proteins Biurette’s test

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To 1ml of the leaves extract, 1ml of 10% sodium hydroxide solution was added and the resulting mixture was heated. To this a drop of 0.7% copper sulphate solution was added. Formation of purplish violet colour indicated the presence of proteins. iii) Test for Steroids Salkowki’s test To 2ml of the leaves extract add 1ml of concentrated sulphuric acid was added carefully along the sides of the test tube. Red colour produced in the chloroform layer indicated the presence of steroids. iv) Test for alkaloids Wagner’s test To 1ml of the leaves extract was acidified by adding 1.5% v/v of HCl and a few drops of Wagner’s reagent. Formation of brown precipitate indicated the presence of alkaloids. v) Test for Flavonoids Zinc hydrochloride test to the leaves extract, a pinch of zinc dust and concentrated HCl were added. Appearance of magenta colour after few minutes indicated the presence of flavonoids. vi) Test for Phenols Lead acetate test 1ml of the leaves extract was diluted with 3 ml of distilled water and to this few drops of 1% aqueous solution of Lead acetate was added. A yellow precipitate was formed which indicated the presence of phenol. vii) Test for Saponins Foam test To the leaves extract a few drops of sodium bicarbonate solution were added. Shaken vigourously and kept for 3 minutes. A honey comb like froth was formed indicated the presence of saponins. viii) Test for Glycosides Legal’s test To the leaves extract, 1ml of sodium nitroprusside solution was added and then it was made alkaline with sodium hydroxide solution. Appearance of pink to red colour confirmed the presence of glycosides. ix) Test for Tannins Lead acetate test To 5ml of the extract add few drops of 1% solution of lead acetate was added. A yellow precipitate was formed indicating the presence of tannins. D) Antibacterial Assay (10) The antibacterial activity was determined by agar well diffusion method. Muller Hinton Agar and Petri plates was sterilized and cooled. Muller Hinton agar (25 ml) was then poured into the sterilized petri plates and allow to solidify. Then 8 hours inoculated young cultures of test organism were spread uniformly with the help of sterile cotton swab on Muller Hinton Agar plates. After that well was made in the plate with the help of sterile cork borer (8 mm) and filled with the leaves extract. For comparative study the well loaded with ethanol serve as control. The plate was then incubated at 370C for 24 hrs. and were observed for zone of inhibition.

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IV. RESULTS AND DISCUSSION From the collected oral samples following bacteria were isolated and identified by gram staining and biochemical test. Bacteria found were Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis and Enterococcus faecalis Table 1: Morphological characteristics of organisms

Organisms Agar Colony Gram Motility Character Character E. coli EMB agar Green metallic Gram –ve Motile sheen P. aeruginosa Cetrimide agar yellow-green Gram –ve Motile colour S. aureus BP agar Black colour Gram +ve Non- Motile B. subtilis Luria-Bertani agar White colour Gram +ve Motile (LB) E. faecalis UTI agar Green colour Gram +ve Motile

Fig 4. E. coli on EMB agar Fig 5. P. aeruginosa on Cetrimide agar

Fig 6. S. aureus on BP agar Fig 7. B. subtilis on LB agar

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Fig 8. E. faecalis on UTI agar

Table 2: Biochemical test- Sugar fermentation test

Sugar Glucose Lactose Mannitol

Organisms Acid Gas Acid Gas Acid Gas E. coli + ve + ve + ve + ve + ve + ve P. aeruginosa + ve - ve - ve - ve - ve - ve S. aureus + ve - ve + ve - ve + ve - ve B. subtilis + ve - ve - ve - ve - ve - ve E. faecalis + ve - ve + ve - ve + ve - ve

Table 3: Biochemical test- IMViC test

Test Indole Methyl Red Voges Proskauer Citrate Coagulase

Organisms E. coli + ve + ve - ve - ve -- P. aeruginosa - ve - ve - ve + ve -- S. aureus - ve + ve + ve - ve +ve

B. subtilis - ve - ve + ve + ve --

E. faecalis - ve - ve + ve - ve --

Fig 9. Biochemical test for E. coli Fig 10. Biochemical test for P. aeruginosa

Fig 11. Biochemical test for S. aureus Fig 12. Biochemical test for B. subtilis

Fig 13. Biochemical test for E. faecalis

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Table 4: Effect of extracts on isolated oral pathogens

Sr. No. Organisms Zone of Zone of Zone of Control inhibition inhibition inhibition (in mm) of J. of J. of J. officinale officinale officinale leaves leaves leaves (aqueous (ethanolic (acetonic extract in extract in extract in mm) mm) mm) 1 E. coli 18 23 13 21 2 P. aeruginosa 12 19 24 20 3 S. aureus 9 17 16 20 4 B. subtilis 10 21 20 15 5 E. faecalis 14 19 25 12

Fig 14. Graphical representation of the effect of J. officinale extracts against ulcer causing oral pathogens. From the above results it was observed that J. officinale give effective result against oral pathogens. In antibacterial activity of leaves of J. officinale extract, the zone of inhibition shown by E. coli was 13mm in Acetone extract, 18mm in Aqueous extract and 23mm in ethanolic extract. The zone of inhibition shown by P. aeruginosa was 12mm in aqueous extract, 24mm in acetone extract and 19mm in ethanolic extract. The zone of inhibition shown by S. aureus was 16mm in Acetone extract, 17mm in Ethanolic extract and 9mm in Aqueous extract. The zone of inhibition shown by B. subtilis was 21mm in Ethanolic extract, 20mm in Acetone extract and 10mm in Aqueous extract. The zone of inhibition shown by E. faecalis was 25mm in Acetone extract, 14mm in Aqueous extract and 19mm in Ethanolic extract.

Fig 15. Antibacterial activity Fig 16. Antibacterial activity shown by E. coli shownDistilled by P.water aeruginosa Ethanol

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Fig 17. Antibacterial activity Fig 18. Antibacterial activity Fig 19. Antibacterial activity shown by S. aureus . shown by B. subtilis shown by E. faecalis

Table 5: Phytochemical analysis

Sr. No. Bioactive Aqueous Ethanolic Acetonic components extract of extract of extract of leaves leaves leaves

1 Carbohydrates - + -

2 Proteins + - + 3 Steroids + - - 4 Alkaloids + + +

5 Flavonoids - + -

6 Phenols + + + 7 Saponins + - - 8 Glycosides + + +

9 Tannins + + +

The crude extracts of leaves of J. officinale indicates the accumulation of alkaloids, saponins, tannins, flavonoids, glycosides and terpenoids. Thus results in present study can be attributed to the presence of these chemical constituents. . DISCUSSION Aqueous extract showed maximum activity against B. subtilis (19 mm), E. coli (17 mm), S. aureus (16 mm), P. aeruginosa (16 mm) and E. faecalis (15 mm). (8) Ethanolic extract showed maximum activity against S. aureus (28.2mm), E. faecalis (15.7mm), E. coli (22.3mm) and P. aeruginosa (23.8mm). (4) The preliminary phytochemical analysis of the aqueous extract of Jasminum officinale leaves indicated the presence of alkaloids, coumarins, flavonoids, tannins, terpenoids, glycosides, emodine, leucoanthcyanins, steroids, anthocyanins, phlobatinins, essential oil and saponins. (2) In the present study, the bacteria found were Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis and Enterococcus faecalis were isolated and identified from collected oral flora of patients. Then antibacterial activity of J. officinale leaves extract in Aqueous, Ethanol and Acetone were analyzed against these isolated oral pathogens.

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On the basis of results, Acetone extract shows maximum inhibition to E. faecalis (25mm), P. aeruginosa (24mm) followed by B. subtilis (20mm), S. aureus (16mm) and E. coli (13mm) Ethanol extract shows maximum inhibition to E. coli (23mm) followed by B. subtilis (21mm), P. aeruginosa (19mm), S. aureus (17mm) and E. faecalis (19mm) Aqueous extract shows maximum inhibition to E. coli (18mm) followed by E. faecalis (14mm), P. aeruginosa (12mm), B. subtilis (10mm) and S. aureus (9mm) V. Conclusion Antibacterial activity of J. officinale leaves extract in Aqueous, Ethanol and Acetone were analyzed against some ulcer causing organisms such as E. coli, P. aeruginosa, S. aureus, B. subtilis and E. faecalis by agar well diffusion assay on Muller Hinton agar media plate. On the basis of results, Acetone extract shows maximum inhibition to E. faecalis, P. aeruginosa followed by B. subtilis, S. aureus and E. coli. Ethanol extract shows maximum inhibition to E. coli followed by B. subtilis, P. aeruginosa, S. aureus and E. faecalis. Aqueous extract shows maximum inhibition to E. coli followed by E. faecalis, P. aeruginosa, B. subtilis and S. aureus. From the above results we can conclude that J. officinale has remarkable antibacterial activity. The medicinal values of the plant leaves may be due to these specific groups of phytochemicals present in it. Phenolic compounds are well known as antioxidant and scavenging agents for free radicals associated with oxidative damage. Tannins are known to be useful in the treatment of inflamed or ulcerated tissues and they have remarkable anticancer property. It has been confirmed that pharmacological effect of flavonoids is also correlating with their antioxidant activities. (11)

Due to these properties of J. officinale, this can be useful in development of new drugs. We can use natural medicines in place of antibiotics in future. VI. References [1] S. Shekhar, M.P. Prasad, “Evaluation of antimicrobial activity of jasminum species using solvent extracts against clinical pathogens,” World Journal of Pharmacy and Pharmaceutical Sciences, Vol. 4(05), 1247-1256, April 2015. [2] A. Al-Snafi, “Pharmacology and Medicinal Properties of Jasminum officinale- A Review,” Indo American Journal of Pharmaceutical Sciences, Vol. 05(04), 2191-2197, January 2018. [3] B. Rani, M. Yadav, G. Pachauri, M. Abid, S. Kazmi, H. Chharang, R.K Maheshwari,. “Awesome medicinal benefits of jasmine plant,” Journal Of Biological And Chemical Research, Vol. 34(2), 918-922, December 2017. [4] Al-Khazraji, S.M.. Evaluation of antibacterial activity of Jasminum officinale, IOSR Journal of Pharmacy and Biological Sciences, Vol. 10(1) II, 2015, 121-124. [5] M. Munoz Corcuera, G. Esparza-Gomez, M.A. Gonzalez-Moles, A. Bascones Martinez, “Oral ulcers: clinical aspects. a tool for dermatologists. part ii. chronic ulcers,” Clin Exp Dermatol, Vol. 34(4), 456-61, 2009. [6] P.S. Subiksha, “Various Remedies for Recurrent Aphthous Ulcer A Review,” Journal of Pharmaceutical Sciences and Research, Vol. 6(6), 251-253, 2014. [7] http://emedicine.medscape.com/article/909213-overview. [8] U. A. Khan, H. Rahman, Z. Niaz, M. Qasim, J. Khan, J. Tayyaba. B. Rehman, “Antibacterial activity of some medicinal plants against selected human pathogenic bacteria, “European Journal of Microbiology and Immunology, Vol. 3(4), 272-274, September 2013. [9] T. Gowdhami, A.K. Rajalakshmi, N. Sugumar, R. Valliappan, “Evaluation of antimicrobial activity of different solvent extracts of aromatic plant: Jasminum sambac linn, “International Journal of Research in Pharmacy and Science, Vol. 5(4) ; 18 –23, December 2015.

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[10] S.M. Al-Khazraji, “Evaluation of antibacterial activity of jasminum officinale,” IOSR Journal of Pharmacy and Biological Sciences, Vol. 10(1) II, 121-124, February 2015. [11] P. Dubey, A. Tiwari, S.K. Gupta, G. Watal, “Phytochemical and Biochemical Studies of Jasminum officinale Leaves,” International Journal of Pharmaceutical Sciences and Research, Vol. 7(6), 2632-2640, March, 2016.

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